Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death

13 pages, 3 tables, 5 figures, http://icm.csic.es/bio/projects/icmicrobis/pdfSince heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Gre ́gori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662–4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano 2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20This work was funded by EU project BASICS (EVK3 CT-2002-00078), Spanish MEC project MODIVUS (CTM2005-04795/MAR), and CIPE research project 17–2003 of the Italian Marche Region. We thank C. Cardelús and V. Balagué for help with the Blanes Bay Microbial Observatory sampling program, L. Alonso-Sáez for sharing the Atlantic data, scientists and technicians on R/V Hesperides for the basic oceanographic data of cruise COCA, and T. Lefort for comments on the manuscript.Peer reviewe